As a means to automate the three-dimensional histological analysis of brain tissue, we demonstrate the use of femtosecond laser pulses to iteratively cut and image fixed as well as fresh tissue. Cuts are accomplished with 1 to 10 uJ pulses to ablate tissue with micron precision. We show that the permeability, immunoreactivity, and optical clarity of the tissue is retained after pulsed laser cutting. Further, samples from transgenic mice that express fluorescent proteins retained their fluorescence to within microns of the cut surface. Imaging of exogenous or endogenous fluorescent labels down to 100um or more below the cut surface is accomplished with 0.1 to 1 nJ pulses and conventional two-photon laser scanning microscopy. In one example, labeled projection neurons within the full extent of a neocortical column were visualized with micron resolution. In a second example, the microvasculature within a block of neocortex was measured and reconstructed with micron resolution.

Bulk heating of transparent materials using a high-repetition-rate femtosecond laser

Femtosecond laser pulses can locally induce structural and chemical changes in the bulk of transparent materials, opening the door to the three-dimensional fabrication of optical devices. We review the laser and focusing parameters that have been applied to induce these changes and discuss the different physical mechanisms that play a role in forming them. We then describe a new technique for inducing refractive-index changes in bulk material using a high-repetition-rate femtosecond oscillator. The changes are caused by a localized melting of the material, which results from an accumulation of thermal energy due to nonlinear absorption of the high-repetition-rate train of laser pulses.

Customization of Poly(dimethylsiloxane) Stamps by Micromachining Using a Femtosecond-Pulsed Laser

This work describes the use of focused, high-intensity light from a Ti:sapphire laser that generates femtosecond pulses to create topographical structure in a flat surface of poly(dimethylsiloxane) (PDMS), and the use of the PDMS surfaces patterned in surface bas-reliefis the material most widely used for printing and stamping in soft lithography, and a material widely used in microfluidic systems. The bas-relief patterns required in these applications are usually fabricated by casting PDMS against a complementary bas-relief pattern in photoresist, fabricated in turn by photolithography. This process works well, but is not applicable to the preparation of PDMS stamps required for all types of problems; printing on spherical surfaces is an example.

Microexplosions in tellurite glasses

Femtosecond laser pulses were used to produce localized damage in the bulk and near the surface of baseline, Al2O3-doped and La2O3-doped sodium tellurite glasses. Single or multiple laser pulses were non-linearly absorbed in the focal volume by the glass, leading to permanent changes in the material in the focal volume. These changes were caused by an explosive expansion of the ionized material in the focal volume into the surrounding material, i.e. a microexplosion. The writing of simple structures (periodic array of voxels, as well as lines) was demonstrated. The regions of microexplosion and writing were subsequently characterized using scanning electron microscopy (SEM), energy-dispersive spectrometry (EDS) and atomic force microscopy (AFM). Fingerprints of microexplosions (concentric lines within the region and a concentric ring outside the region), due to the shock wave generated during microexplosions, were evident. In the case of the baseline glass, no chemistry change was observed within the region of the microexplosion. However, Al2O3-doped and La2O3-doped glasses showed depletion of the dopant fromthe edge to the center of the region of the microexplosions, indicating a chemistry gradient within the regions. Interrogation of the bulk- and lasertreated regions using micro-Raman spectroscopy revealed no structural change due to the microexplosions and writing within these glasses